The present invention relates to a hyperthermostable protease useful as an enzyme for industrial use, a gene encoding the same and a method of producing the enzyme by genetic engineering technique.
A protease is an enzyme that cleaves peptide bonds in proteins. A number of such enzymes have been found in animals, plants and microorganisms. The protease is used as a reagent for laboratory use and as a pharmaceutical, as well as in industrial fields, for example, as an additive for a detergent, for processing foods and for chemical synthesis utilizing a reverse reaction. Therefore, it can be said that the protease is an extremely important enzyme for industries. Since high physical and chemical stability is required for a protease used in industrial fields, a thermostable enzyme is preferably used among others. Since proteases produced by bacteria of genus Bacillus exhibit relatively high thermostability, they are mainly used as proteases for industrial use. However, in search of a more superior enzyme, attempts have been made to obtain an enzyme from a microorganism growing at high temperature, for example, a thermophilic bacterium of genus Bacillus or a hyperthermophile.
For example, a hyperthermophile Pyrococcus furiosus is known to produce a protease (Appl. Environ. Microbiol., 56:1992-1998 (1990); FEMS Microbiol. Letters, 71:17-20 (1990); J. Gen. Microbiol., 137:1193-1199 (1991)).
In addition, a hyperthermophile, Pyrococcus sp. strain KOD1, is reported to produce a thiol protease (a cysteine protease) (Appl. Environ. Microbiol., 60:4559-4566 (1994)). Hyperthermophiles of genus Thermococcus, genus Staphylothermus and genus Thermobacteroides are also known to produce proteases (Appl. Microbiol. Biotechnol., 34:715-719 (1991)).
The proteases from the hyperthermophiles as described above have high thermostability. Therefore, it is expected that they may be used in place of the thermostable proteases currently in use or in a field in which use of a protease has not been considered.
However, most of the microorganisms producing these enzymes grow only at high temperature. For example, Pyrococcus furiosus needs to be cultured at 90-100xc2x0 C. Culturing at such high temperature is disadvantageous in view of energy cost. Furthermore, the productivities of the proteases from the hyperthermophiles are lower than the productivities of the conventional microbial proteases. Thus, the methods for industrially producing the proteases from the hyperthermophiles have problems.
By the way, production of an enzyme by genetic engineering technique by isolating the gene for the enzyme of interest and introducing it into a host microorganism that can readily be cultured is currently common in the art. However, the gene for the enzyme introduced into the host is not always expressed so efficiently as expected. It is believed that the main cause is that the GC content or the codon usage of the introduced gene is different from those of the genes of the host. Therefore, it is necessary to optimize the expression method for each gene to be introduced and/or each host in order to accomplish a suitable productivity of an enzyme for the intended use.
The objects of the present invention are to provide a protease from a hyperthermophile which is advantageous for industrial use, to isolate a gene encoding the protease from the hyperthermophile, and to provide a method of producing the hyperthermostable protease using the gene by genetic engineering technique in order to solve the problems as described above.
Among proteases produced by hyperthermophiles, some may be classified into the subtilisin-type of alkaline proteases based on the amino acid sequence homology. When a gene for such a protease is introduced into Bacillus subtilis which is generally used for production by genetic engineering technique, the productivity of this enzyme is much less than that of a protein inherently produced by Bacillus subtilis. 
The present inventors have studied intensively and found that, by placing a gene encoding a signal peptide (signal sequence) derived from a subtilisin upstream a protease gene derived from a hyperthermophile to be expressed, and modifying the amino acid sequence around the cleavage site, the gene of interest is expressed in Bacillus subtilis with high efficiency. Furthermore, it has been found that the expression level of the enzyme can be increased by deleting a portion that is not essential for the enzymatic activity in the protease gene derived from the hyperthermophile of interest. Thus, the present invention has been completed.
The present invention is outlined as follows. The first invention of the present invention is a thermostable protease having an amino acid sequence represented by the SEQ ID NO:1 of the Sequence Listing, and a protease having an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted or added in the amino acid sequence represented by the SEQ ID NO:1 of the Sequence Listing and having a thermostable protease activity.
The second invention of the present invention is a gene encoding the thermostable protease of the first invention, and a thermostable protease gene that hybridizes with the gene.
The third invention of the present invention is a gene to be used for producing a thermostable protease derived from a hyperthermophile by genetic engineering technique, characterized in that the gene encodes an amino acid sequence represented by formula I:
SIG-Ala-Gly-Gly-Asn-PRO [I] (SEQ ID NO:30)
wherein SIG represents an amino acid sequence of a signal peptide derived from a subtilisin, PRO represents an amino acid sequence of a protein to be expressed. Preferably, SIG is the amino acid sequence represented by the SEQ ID NO:3 of the Sequence Listing. Preferably, PRO is an amino acid sequence of a hyperthermostable protease derived from a hyperthermophile, more preferably, an amino acid sequence of a protease derived from Pyrococcus furiosus. 
The fourth invention of the present invention relates to a method of producing a protein by genetic engineering technique, characterized in that the method comprises culturing a bacterium of genus Bacillus into which the gene of the third invention is introduced, and collecting the protein of interest from the culture.
The fifth invention of the present invention is a plasmid used for producing a protein by genetic engineering technique, characterized in that the gene of the third invention is inserted into the plasmid.
A mutation such as deletion, substitution, insertion or addition of one to several amino acid residues in an amino acid sequence may be generated in a naturally occurring protein including the protein disclosed by the present invention. Such mutation may be generated due to a polymorphism or a mutation of the gene encoding the protein, or due to a modification of the protein in vivo or during purification after synthesis may occur. Nevertheless, it is known that such a mutated protein may exhibit physiological and biological activities equivalent with those of a protein without a mutation. This is applicable to a protein in which such a mutation is introduced into its amino sequence artificially, in which case it is possible to generate a wide variety of mutations. For example, it is known that a polypeptide in which a cysteine residue in the amino acid sequence of human interleukin-2 (IL-2) is substituted with a serine residue retains an interleukin-2 activity (Science, 224:1431 (1984)). Thus, a protease having an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted or added in the amino acid sequence disclosed by the present invention and having a protease activity equivalent with that of the protease of the present invention is within the scope of the present invention.
As used herein, xe2x80x9ca gene which hybridizes (with a particular gene)xe2x80x9d is a gene having a base sequence similar to that of the particular gene. It is likely that a gene having a base sequence similar to that of a particular gene encodes a protein having an amino acid sequence and a function similar to those of the protein encoded by the particular gene. Similarity of base sequences of genes can be examined by determining whether or not the genes or portions thereof form a hybrid (hybridize) each other under stringent conditions. By utilizing this procedure, a gene that encodes a protein having a similar function with that of the protein encoded by the particular gene can be obtained. That is, a gene having a similar base sequence with that of the gene of the present invention can be obtained by using the gene obtained by the present invention or a portion thereof as a probe to carry out hybridization according to a known method. Hybridization can be carried out according to the method, for example, as described in T. Maniatis et al. eds., Molecular Cloning: A Laboratory Manual 2nd ed., published by Cold Spring Harbor Laboratory, 1989. More specifically, hybridization can be carried out under the following conditions. Briefly, a membrane onto which DNAs are immobilized is incubated in 6xc3x97SSC (1xc3x97SSC represents 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) containing 0.5% SDS, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrorridone, 0.1% Ficoll 400, 0.01% denatured salmon sperm DNA at 50xc2x0 C. for 12-20 hours with a probe. After incubation, the membrane is washed until the signals for the immobilized DNAs can be distinguished from background, starting from washing in 2xc3x97SSC containing 0.5% SDS at 37xc2x0 C. while decreasing the SSC concentration down to 0.1xc3x97 and raising the temperature up to 50xc2x0 C.
Alternatively, instead of hybridization, a gene amplification method (e.g., PCR method) which employs portions of the base sequence of the gene obtained by the present invention as primers can be utilized. Whether or not the gene thus obtained encodes a protein having the function of interest can be determined by expressing the gene utilizing a suitable host and a suitable expression system and examining the activity of the resulting protein.